In silico design of DNA sequences for in vivo nucleosome positioning.

The computational design of synthetic DNA sequences with designer in vivo properties is gaining traction in the field of synthetic genomics. We propose here a computational method which combines a kinetic Monte Carlo framework with a deep mutational screening based on deep learning predictions. We apply our method to build regular nucleosome arrays with tailored nucleosomal repeat lengths (NRL) in yeast. Our design was validated in vivo by successfully engineering and integrating thousands of kilobases long tandem arrays of computationally optimized sequences which could accommodate NRLs much larger than the yeast natural NRL (namely 197 and 237 bp, compared to the natural NRL of ∼165 bp). RNA-seq results show that transcription of the arrays can occur but is not driven by the NRL. The computational method proposed here delineates the key sequence rules for nucleosome positioning in yeast and should be easily applicable to other sequence properties and other genomes.

Stable structures or PABP1 loading protects cellular and viral RNAs against ISG20-mediated decay.

ISG20 is an IFN-induced 3'-5' RNA exonuclease that acts as a broad antiviral factor. At present, the features that expose RNA to ISG20 remain unclear, although recent studies have pointed to the modulatory role of epitranscriptomic modifications in the susceptibility of target RNAs to ISG20. These findings raise the question as to how cellular RNAs, on which these modifications are abundant, cope with ISG20. To obtain an unbiased perspective on this topic, we used RNA-seq and biochemical assays to identify elements that regulate the behavior of RNAs against ISG20. RNA-seq analyses not only indicate a general preservation of the cell transcriptome, but they also highlight a small, but detectable, decrease in the levels of histone mRNAs. Contrarily to all other cellular ones, histone mRNAs are non-polyadenylated and possess a short stem-loop at their 3' end, prompting us to examine the relationship between these features and ISG20 degradation. The results we have obtained indicate that poly(A)-binding protein loading on the RNA 3' tail provides a primal protection against ISG20, easily explaining the overall protection of cellular mRNAs observed by RNA-seq. Terminal stem-loop RNA structures have been associated with ISG20 protection before. Here, we re-examined this question and found that the balance between resistance and susceptibility to ISG20 depends on their thermodynamic stability. These results shed new light on the complex interplay that regulates the susceptibility of different classes of viruses against ISG20.

Distinct evolutionary trajectories of SARS-CoV-2-interacting proteins in bats and primates identify important host determinants of COVID-19.

The coronavirus disease 19 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a coronavirus that spilled over from the bat reservoir. Despite numerous clinical trials and vaccines, the burden remains immense, and the host determinants of SARS-CoV-2 susceptibility and COVID-19 severity remain largely unknown. Signatures of positive selection detected by comparative functional genetic analyses in primate and bat genomes can uncover important and specific adaptations that occurred at virus-host interfaces. We performed high-throughput evolutionary analyses of 334 SARS-CoV-2-interacting proteins to identify SARS-CoV adaptive loci and uncover functional differences between modern humans, primates, and bats. Using DGINN (Detection of Genetic INNovation), we identified 38 bat and 81 primate proteins with marks of positive selection. Seventeen genes, including the ACE2 receptor, present adaptive marks in both mammalian orders, suggesting common virus-host interfaces and past epidemics of coronaviruses shaping their genomes. Yet, 84 genes presented distinct adaptations in bats and primates. Notably, residues involved in ubiquitination and phosphorylation of the inflammatory RIPK1 have rapidly evolved in bats but not primates, suggesting different inflammation regulation versus humans. Furthermore, we discovered residues with typical virus-host arms race marks in primates, such as in the entry factor TMPRSS2 or the autophagy adaptor FYCO1, pointing to host-specific in vivo interfaces that may be drug targets. Finally, we found that FYCO1 sites under adaptation in primates are those associated with severe COVID-19, supporting their importance in pathogenesis and replication. Overall, we identified adaptations involved in SARS-CoV-2 infection in bats and primates, enlightening modern genetic determinants of virus susceptibility and severity.

Chromosome-level genome assembly reveals homologous chromosomes and recombination in asexual rotifer Adineta vaga.

Bdelloid rotifers are notorious as a speciose ancient clade comprising only asexual lineages. Thanks to their ability to repair highly fragmented DNA, most bdelloid species also withstand complete desiccation and ionizing radiation. Producing a well-assembled reference genome is a critical step to developing an understanding of the effects of long-term asexuality and DNA breakage on genome evolution. To this end, we present the first high-quality chromosome-level genome assemblies for the bdelloid Adineta vaga, composed of six pairs of homologous (diploid) chromosomes with a footprint of paleotetraploidy. The observed large-scale losses of heterozygosity are signatures of recombination between homologous chromosomes, either during mitotic DNA double-strand break repair or when resolving programmed DNA breaks during a modified meiosis. Dynamic subtelomeric regions harbor more structural diversity (e.g., chromosome rearrangements, transposable elements, and haplotypic divergence). Our results trigger the reappraisal of potential meiotic processes in bdelloid rotifers and help unravel the factors underlying their long-term asexual evolutionary success.

Effects of bioturbation by tubificid worms on biogeochemical processes, bacterial community structure and diversity in heterotrophic wetland sediments.

Bioturbation activity of tubificid worms has been recognized as a key process influencing organic matter processing and nutrient cycling in benthic aquatic ecosystems. This activity is expected to modify benthic microbial communities by affecting the physical and chemical environment in sediments. Nevertheless, quantifications of bacterial community changes associated with bioturbation in freshwater ecosystems are still lacking. The present study aimed at evaluating the impact of tubificid worms on bacterial community structure using NGS approach (16S metabarcoding) and long (6 months) laboratory experiments on four heterotrophic wetland sediments. Worm bioturbation activity significantly stimulated biogeochemical processes at the water-sediment interface but only had a marginally significant effect on bacterial community structures. Yet, bacterial diversity was consistently reduced in presence of worms. Such decrease could be associated with the stimulation of organic matter mineralization by worms, leading to a reduction of the diversity of trophic niches available for bacterial species. The slight changes in bacterial community structures induced by bioturbation did not appear to control biogeochemical processes. Thus, the stimulation of biogeochemical processes by worm bioturbation was more associated with a stimulation of the initial bacterial community than with a drastic change in bacterial communities induced by worms.

First Case of Brucellosis Caused by an Amphibian-type Brucella.

We report the first case of brucellosis caused by an isolate whose genome is identical that of a frog isolate from Texas, demonstrating the zoonotic potential of amphibian-type Brucella. Importantly, with such atypical Brucella, correct diagnosis cannot be performed using routine serological tests or identification methods.

A prospective survey of Streptococcus pyogenes infections in French Brittany from 2009 to 2017: Comprehensive dynamic of new emergent emm genotypes.

Streptococcus pyogenes or group A Streptococcus (GAS) causes diseases ranging from uncomplicated pharyngitis to life-threatening infections. It has complex epidemiology driven by the diversity, the temporal and geographical fluctuations of the circulating strains. Despite the global burden of GAS diseases, there is currently no available vaccination strategy against GAS infections. This study, based on a longitudinal population survey, aimed to understand the dynamic of GAS emm types and to give leads to better recognition of underlying mechanisms for the emergence of successful clones. From 2009 to 2017, we conducted a systematic culture-based diagnosis of GAS infections in a French Brittany population with a prospective recovery of clinical data. The epidemiological analysis was performed using emm typing combined with the structural and functional cluster-typing system for all the recovered strains. Risk factors for the invasiveness, identified by univariate analysis, were computed in a multiple logistic regression analysis, and the only independent risk factor remaining in the model was the age (OR for the entire range [CI95%] = 6.35 [3.63, 11.10]; p<0.0001). Among the 61 different emm types identified, the most prevalent were emm28 (16%), emm89 (15%), emm1 (14%), and emm4 (8%), which accounted for more than 50% of circulating strains. During the study period, five genotypes identified as emm44, 66, 75, 83, 87 emerged successively and belonged to clusters D4, E2, E3, and E6 that were different from those gathering "Prevalent" emm types (clusters A-C3 to 5, E1 and E4). We previously reported significant genetic modifications for emm44, 66, 83 and 75 types resulting possibly from a short adaptive evolution. Herein we additionally observed that the emergence of a new genotype could occur in a susceptible population having specific risk factors or probably lacking a naturally-acquired cluster-specific immune cross-protection. Among emergent emm types, emm75 and emm87 tend to become prevalent with a stable annual incidence and the risk of a clonal expansion have to be considered.

How consistent is RAD-seq divergence with DNA-barcode based clustering in insects?

Promoted by the barcoding approach, mitochondrial DNA is more than ever used as a molecular marker to identify species boundaries. Yet, it has been repeatedly argued that it may be poorly suited for this purpose, especially in insects where mitochondria are often associated with invasive intracellular bacteria that may promote their introgression. Here, we inform this debate by assessing how divergent nuclear genomes can be when mitochondrial barcodes indicate very high proximity. To this end, we obtained RAD-seq data from 92 barcode-based species-like units (operational taxonomic units [OTUs]) spanning four insect orders. In 100% of the cases, the observed median nuclear divergence was lower than 2%, a value that was recently estimated as one below which nuclear gene flow is not uncommon. These results suggest that although mitochondria may occasionally leak between species, this process is rare enough in insects to make DNA barcoding a reliable tool for clustering specimens into species-like units.

Comparing 16S rDNA amplicon sequencing and hybridization capture for pea aphid microbiota diversity analysis.

OBJECTIVE: Targeted sequencing of 16S rDNA amplicons is routinely used for microbial community profiling but this method suffers several limitations such as bias affinity of universal primers and short read size. Gene capture by hybridization represents a promising alternative. Here we used a metagenomic extract from the pea aphid Acyrthosiphon pisum to compare the performances of two widely used PCR primer pairs with DNA capture, based on solution hybrid selection. RESULTS: All methods produced an exhaustive description of the 8 bacterial taxa known to be present in this sample. In addition, the methods yielded similar quantitative results, with the number of reads strongly correlating with quantitative PCR controls. Both methods can thus be considered as qualitatively and quantitatively robust on such a sample with low microbial complexity.

Emergence of Nontuberculous Mycobacterial Lymphadenitis in Children After the Discontinuation of Mandatory Bacillus Calmette and GuÉrin Immunization in France.

Eighty-five children were diagnosed with culture-confirmed nontuberculous mycobacterial cervical lymphadenitis within the MYCOMED surveillance network from 2004 to 2013. The mean incidence sharply increased from 0.57 to 3.7 per 100,000 children per year, after the discontinuation of mandatory bacillus Calmette and Guérin immunization in 2007. Cases were documented as Mycobacterium avium (62.3%), Mycobacterium intracellulare (15.3%) and Mycobacterium lentiflavum (12.9%). Outcome was favorable in all, with or without surgery or antimycobacterial treatment.

How and how much does RAD-seq bias genetic diversity estimates?

BACKGROUND: RAD-seq is a powerful tool, increasingly used in population genomics. However, earlier studies have raised red flags regarding possible biases associated with this technique. In particular, polymorphism on restriction sites results in preferential sampling of closely related haplotypes, so that RAD data tends to underestimate genetic diversity. RESULTS: Here we (1) clarify the theoretical basis of this bias, highlighting the potential confounding effects of population structure and selection, (2) confront predictions to real data from in silico digestion of full genomes and (3) provide a proof of concept toward an ABC-based correction of the RAD-seq bias. Under a neutral and panmictic model, we confirm the previously established relationship between the true polymorphism and its RAD-based estimation, showing a more pronounced bias when polymorphism is high. Using more elaborate models, we show that selection, resulting in heterogeneous levels of polymorphism along the genome, exacerbates the bias and leads to a more pronounced underestimation. On the contrary, spatial genetic structure tends to reduce the bias. We confront the neutral and panmictic model to "ideal" empirical data (in silico RAD-sequencing) using full genomes from natural populations of the fruit fly Drosophila melanogaster and the fungus Shizophyllum commune, harbouring respectively moderate and high genetic diversity. In D. melanogaster, predictions fit the model, but the small difference between the true and RAD polymorphism makes this comparison insensitive to deviations from the model. In the highly polymorphic fungus, the model captures a large part of the bias but makes inaccurate predictions. Accordingly, ABC corrections based on this model improve the estimations, albeit with some imprecisions. CONCLUSION: The RAD-seq underestimation of genetic diversity associated with polymorphism in restriction sites becomes more pronounced when polymorphism is high. In practice, this means that in many systems where polymorphism does not exceed 2 %, the bias is of minor importance in the face of other sources of uncertainty, such as heterogeneous bases composition or technical artefacts. The neutral panmictic model provides a practical mean to correct the bias through ABC, albeit with some imprecisions. More elaborate ABC methods might integrate additional parameters, such as population structure and selection, but their opposite effects could hinder accurate corrections.

An original case of Francisella tularensis subsp. holarctica bacteremia after a near-drowning accident.

We report the first case of Francisella tularensis subsp. holarctica bacteremia after water contamination in France. A 75-year-old man developed septic pneumonic tularemia after a near-drowning accident. We highlight the need for a longer incubation time for isolation of F. tularensis from blood cultures.

Chemical and morphological filters in a specialized floral mimicry system.

Many plant species attract insect pollinators through chemical mimicry of their oviposition sites, often detaining them in a trap chamber that ensures pollen transfer. These plant mimics are considered to be unspecialized at the pollinator species level, yet field observations of a mycoheterotrophic rainforest orchid (Gastrodia similis), which emits an odour reminiscent of rotting fruit, indicate that it is pollinated by a single drosophilid fly species (Scaptodrosophila bangi). We investigated the roles of floral volatiles and the dimensions of the trap chamber in enforcing this specialization, using gas chromatography-mass spectrometry analyses, bioassays and scanning electron microscopy. We showed that G. similis flowers predominantly emit three fatty-acid esters (ethyl acetate, ethyl isobutyrate and methyl isobutyrate) that were shown in experiments to attract only Scaptodrosophila flies. We additionally showed that the trap chamber, which flies enter into via a touch-sensitive 'trapdoor', closely matches the body size of the pollinator species S. bangi and plays a key role in pollen transfer. Our study demonstrates that specialization in oviposition site mimicry is due primarily to volatile chemistry and is reflected in the dimensions of the trapping apparatus. It also indicates that mycoheterotrophic plants can be specialized both on mycorrhizal fungi and insect pollinators.

Optimization of multiplexed RADseq libraries using low-cost adaptors.

Reduced representation genomics approaches, of which RADseq is currently the most popular form, offer the possibility to produce genome wide data from potentially any species, without previous genomic information. The application of RADseq to highly multiplexed libraries (including numerous specimens, and potentially numerous different species) is however limited by technical constraints. First, the cost of synthesis of Illumina adaptors including molecular identifiers (MIDs) becomes excessive when numerous specimens are to be multiplexed. Second, the necessity to empirically adjust the ratio of adaptors to genomic DNA concentration impedes the high throughput application of RADseq to heterogeneous samples, of variable DNA concentration and quality. In an attempt to solve these problems, we propose here some adjustments regarding the adaptor synthesis. First, we show that the common and unique (MID) parts of adaptors can be synthesized separately and subsequently ligated, which drastically reduces the synthesis cost, and thus allows multiplexing hundreds of specimens. Second, we show that self-ligation of adaptors, which makes the adaptor concentration so critical, can be simply prevented by using unphosphorylated adaptors, which significantly improves the ligation and sequencing yield.

Full-Length Genome Sequence of Type M/emm83 Group A Streptococcus pyogenes Strain STAB1101, Isolated from Clustered Cases in Brittany.

Here, we announce the complete annotated genome sequence of a Streptococcus pyogenes M/emm83 strain, STAB1101, isolated from clustered cases in homeless persons in Brittany (France). The genome is composed of 1,709,790 bp, with a G+C content of 38.4% and 1,550 identified coding sequences (CDS), and it harbors a Tn916-like transposon.

Distinguishing migration from isolation using genes with intragenic recombination: detecting introgression in the Drosophila simulans species complex.

BACKGROUND: Determining the presence or absence of gene flow between populations is the target of some statistical methods in population genetics. Until recently, these methods either avoided the use of recombining genes, or treated recombination as a nuisance parameter. However, genes with recombination contribute additional information for the detection of gene flow (i.e. through linkage disequilibrium). METHODS: We present three summary statistics based on the spatial arrangement of fixed differences, and shared and exclusive polymorphisms that are sensitive to the presence and direction of gene flow. Power and false positive rate for tests based on these statistics are studied by simulation. RESULTS: The application of these tests to populations from the Drosophila simulans species complex yielded results consistent with migration between D. simulans and its two endemic sister species D. mauritiana and D. sechellia, and between populations D. mauritiana on the islands of the Mauritius and Rodrigues. CONCLUSIONS: We demonstrate the sensitivity of the developed statistics to the presence and direction of gene flow, and characterize their power as a function of differentiation level and recombination rate. The properties of these statistics make them especially suitable for analyzing high-throughput sequencing data or for their integration within the approximate Bayesian computation framework.

Is RAD-seq suitable for phylogenetic inference? An in silico assessment and optimization.

INFERRING PHYLOGENETIC RELATIONSHIPS BETWEEN CLOSELY RELATED TAXA CAN BE HINDERED BY THREE FACTORS: (1) the lack of informative molecular variation at short evolutionary timescale; (2) the lack of established markers in poorly studied taxa; and (3) the potential phylogenetic conflicts among different genomic regions due to incomplete lineage sorting or introgression. In this context, Restriction site Associated DNA sequencing (RAD-seq) seems promising as this technique can generate sequence data from numerous DNA fragments scattered throughout the genome, from a large number of samples, and without preliminary knowledge on the taxa under study. However, divergence beyond the within-species level will necessarily reduce the number of conserved and non-duplicated restriction sites, and therefore the number of loci usable for phylogenetic inference. Here, we assess the suitability of RAD-seq for phylogeny using a simulated experiment on the 12 Drosophila genomes, with divergence times ranging from 5 to 63 million years. These simulations show that RAD-seq allows the recovery of the known Drosophila phylogeny with strong statistical support, even for relatively ancient nodes. Notably, this conclusion is robust to the potentially confounding effects of sequencing errors, heterozygosity, and low coverage. We further show that clustering RAD-seq data using the BLASTN and SiLiX programs significantly improves the recovery of orthologous RAD loci compared with previously proposed approaches, especially for distantly related species. This study therefore validates the view that RAD sequencing is a powerful tool for phylogenetic inference.

[Vancomycin-induced DRESS syndrome: a case report]. / DRESS (Drug reaction with eosinophilia and systemic symptoms) suite à la prise de vancomycine : à propos d'un cas.

DRESS syndrome is a severe adverse drug-induced reaction, characterized by generalized skin rash associated with hypereosinophilia, lymphocytosis and internal organ involvement. Antiepileptics, sulfamides and allopurinol are the most frequently reported drugs; vancomycin is less common. We report a case of vancomycin-induced DRESS syndrome in a 69-year-old male patient. Clinical symptoms and diagnosis difficulties are reported through this observation as well as pathogenesis and treatment of this syndrom.

Microsatellite variation suggests a recent fine-scale population structure of Drosophila sechellia, a species endemic of the Seychelles archipelago.

Drosophila sechellia is closely related to the cosmopolitan and widespread model species, D. simulans. This species, endemic to the Seychelles archipelago, is specialized on the fruits of Morinda citrifolia, and harbours the lowest overall genetic diversity compared to other species of Drosophila. This low diversity is associated with a small population size. In addition, no obvious population structure has been evidenced so far across islands of the Seychelles archipelago. Here, a microsatellite panel of 17 loci in ten populations from nine islands of the Seychelles was used to assess the effect of the D. sechellia's fragmented distribution on the fine-scale population genetic structure, the migration pattern, as well as on the demography of the species. Contrary to previous results, also based on microsatellites, no evidence for population contraction in D. sechellia was found. The results confirm previous studies based on gene sequence polymorphism that showed a long-term stable population size for this species. Interestingly, a pattern of Isolation By Distance which had not been described yet in D. sechellia was found, with evidence of first-generation migrants between some neighbouring islands. Bayesian structuring algorithm results were consistent with a split of D. sechellia into two main groups of populations: Silhouette/Mahé versus all the other islands. Thus, microsatellites suggest that variability in D. sechellia is most likely explained by local genetic exchanges between neighbouring islands that have recently resulted in slight differentiation of the two largest island populations from all the others.